Neuron Like Differentiation of Bone Marrow Derived Mesenchymal Stem Cells

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Neuron-Like Differentiation of Bone Marrow-Derived

Mesenchymal Stem Cells

Keum Seok Bae,

1

Joon Beom Park,

1

Hyun Soo Kim,

2,4

Dae Sung Kim,

1

Dong Jun Park,

3

and Seong Joon Kang

1

Departments of 1Surgery, 2Hemato-Oncology and 3Otorhinolaryngology, Yonsei University Wonju College of Medicine, Wonju, Korea; 4FCB-Pharmicell Co., Ltd. Seongnam, Korea.

Received: April 17, 2010 Revised: July 25, 2010 Accepted: July 26, 2010

Corresponding author: Dr. Seong Joon Kang, Department Surgery, Yonsei University Wonju College of Medicine, 162 Ilsan-dong, Wonju 220-701, Korea.

Tel: 82-33-741-1306, Fax: 82-33-742-1815 E-mail: mdkang@yonsei.ac.kr

∙ The authors have no financial conflicts of interest.

© Copyright:

Yonsei University College of Medicine 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Purpose: Mesenchymal stem cells (MSCs) are multipotent and give rise to distinct-ly differentiated cells from all three germ layers. Neuronal differentiation of MSC has great potential for cellular therapy. We examined whether the cluster of mechani-cally made, not neurosphere, could be differentiated into neuron-like cells by growth factors, such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). Materials and Methods:BMSCs grown confluent were mechanically separated with cell scrapers and masses of sepa-rated cells were cultured to form cluster BMSCs. As described here cluster of BM-SCs were differentiated into neuron-like cells by EGF, HGF, and VEGF. Differenti-ated cells were analyzed by means of phase-contrast inverted microscopy, reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, and immu-nocytochemistry to identify the expression of neural specific markers. Results: For the group with growth factors, the shapes of neuron-like cells was observable a week later, and two weeks later, most cells were similar in shape to neuron-like cells. Par-ticularly, in the group with chemical addition, various shapes of filament structures were seen among the cells. These culture conditions induced MSCs to exhibit a neu-ral cell phenotype, expressing seveneu-ral neuro-glial specific markers. Conclusion:

bone marrow-derived mesenchymal stem cells (BMSCs) could be easily induced to form clusters using mechanical scraping, not neurospheres, which in turn could dif-ferentiate further into neuron-like cells and might open an attractive possibility for clinical cell therapy for neurodegenerative diseases. In the future, we consider that neuron-like cells differentiated from clusters of BMSCs are needed to be compared and analyzed on a physiological and molecular biological level with preexisting neu-ronal cells, and studies on the possibility of their transplantation and differentiation capability in animal models are further required.

Key Words: Neuron-like cells, mesenchymal stem cell, epidermal growth factor, vascular endothelial growth factor, hepatocyte growth factor

INTRODUCTION

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non-into a blood vessel in a bone marrow transplantation model.7

Mezey, et al.8 also discovered that when bone marrow cells

are injected into mouse peritoneum, these cells migrate to the brain and differentiate into neurons.

Recent studies also report that these marrow-derived mes-enchymal cells have a capacity to differentiate into neurons in ex vivo surroundings. Sanchez-Ramos, et al.20 used

retino-ic acid and brain-derived neurotrophretino-ic factors to differentiate BMSCs into neural cells, including neurons and astrocytes. Woodbury, et al.21 determined that using antioxidants such as

β-mercaptoethanol (BME), dimethyl sulfoxide (DMSO), and butylated hydroxyanisole (BHA), they differentiated mar-row-derived mesenchymal cells into neurons. The result was that within a few hours, most MSCs (80%) transformed into neuron-like shapes and expressed neuron-specific markers.

In the studies mentioned above concerning the potential of BMSC differentiation into neuron-like cells, the results from these studies suggest that these MSCs can be applied to the treatment of various brain and nerve diseases. There-fore, the goal of this study is to determine an effective meth-od of differentiating BMSCs into neuron-like cells. We used EGF, vascular endothelial growth factor (VEGF), and hepa-tocyte growth factor (HGF), and observed the conditions of differentiation.

EGF and VEGF are known to function as growth factors that stimulate proliferation of brain tissue-derived neural mesenchymal cells in culture.22-26 HGF is a heterodimer that

consists of a chain containing four kringles and a serine protease-like b chain and is also called as scatter factor. It binds to c-Met, the tyrosine kinase receptor,27 and it is a

growth factor that has pleiotrophic roles.28 Above all, within

the neural tissue, tyrosine kinase receptor and its ligands are expressed, and they play an important role in survival, dif-ferentiation and regeneration of neurons.

The ability of these cells to neurogenically differentiate has deep potential that is applicable to the field of cell ther-apy. Nerve tissue has a limited ability to repair itself after injury.29-32 Generally, groups of cells suggested for

neuro-genic cell therapy are embryonic stem cells and neuron stem cells acquired in embryo or adult brain tissue.33-36 However,

the use of these cells has limited application in clinical cir-cumstances due to ethical and legal issues.

BMSCs and their ability to differentiate into neuron-like cells play an important role in the treatment of degenerative nerve diseases such as Parkinson’s disease, Huntington’s disease, and Amyotrophic Lateral sclerosis.37-39 It is being

recognized that bone marrow that functions as storage for hematopoietic stem cells besides the most common stem

cells of hematopoietic origin. In 1968, Friedenstein et al proved that these cells can be formed into bone, and in 1985 Owen1 reported that cells with self renewal and

multi-differentiation capacity may be present in bone marrow on the basis of these cells’ similarity to hematopoietic cells. These have been named as bone marrow derived mesen-chymal stem cells (MSCs), or marrow stromal stem cells, and there are many researches who focus their aims on these cells.

MSCs are harvested from fat, cord blood, and embryos as well as from bone marrow. They have the potential to dif-ferentiate into marrow stromal cells, fat cells, osteoblastic cells, chondrocytes, tendinocytes, and myocytes, which are normally derived from mesenchymal stem cells.2-13 They

are also capable of differentiating into endodermal origin hepatocytes and ectodermal origin neurons. Therefore, these differentiation potentials of MSCsare used for research into possible clinical utilization in regenerative cell therapy.

Generally, the number of MSCs in the bone marrow is small. There are about 2 to 5 cells present in every 1×106

mononuclear cells. Normally, the human body is thought to have about 1×106 MSCs. However, they are relatively

easi-ly separated and grown in culture, and they have a great proliferation capability of a billion-fold increase in number

ex vivo without losing their stem cell characteristics. Nota-bly, Kuznetsov, et al.14 identified several growth factors that

are related to MSCs. They reported that MSCs under the presence of serum, platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), and epidermal growth factor (EGF) can form in vitro colonies.15

The Ancient Chinese once said that the “brain is the sea of bone marrow.” These ancient beliefs are now being prov-en one by one in recprov-ent times.16 Eglitis and Mezey17

report-ed bone marrow derivreport-ed cells discoverreport-ed in the all brain parts from the cortex to the brain stem. Particularly, in the case of human MSCs, they migrate and survive similarly to mice astrocytes when grafted in mice striatum, and they are discovered to lose their marrow mesenchymal cell mark-ers.18 When marrow-derived mesenchymal cells are

trans-planted into the lateral ventricles of a mouse, Kopen, et al.19

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ROTEC), CD73-PE (BD), CD45-FITC (BD), CD34-FITC (BD), CD14-FITC (BD), and control (IgG1-FITC and IgG1-PE, BD)] for 45 min at room temperature. They were then washed three times with PBS and precipitated. The cells were dripped into columns and fixed with flow buffer (0.1% sodium azide, 1% paraformaldehyde, 0.5% bovine serum albumin). Finally, the cells were analyzed under Ep-ics XL (Beckman Coulter, Brea, CA, USA).

Differentiation capability test

Osteogenic differentiation of BMSCs

BMSCs (second passage) in the early phase of culture were suspended to DMEM. They were divided into 24 well plates (5×104 cells/well) and cultured for 24 hours. Then, the

me-dium was replaced with osteoblast differentiation meme-dium [High-glucose DMEM, 10% fetal bovine serum, 0.1 µM dexamethasone (Sigma, St. Louis, MO, USA), 10 mM β-glycerol phosphate, 50 µM L-ascorbic acid] and cultured for three weeks. The medium was replaced twice a week.

Chondrogenic differentiation of BMSCs

BMSCs were suspended in chondrogenic medium (6.25 μg/mL insulin, 6.25 μg/mL transferrin, 6.25 μg/mL sele-nous acid, 5.35 μg/mL linoleic acid, 1.25 μg/mL bovine se-rum albumin), 1 mM Pyruvate, 37.5 μg/mL Ascorbic acid, 10-7 M dexamethasone (Sigma)]. It was then centrifuged for 10 minutes in 450 xg. The precipitated cells were cul-tured in chondrogenic medium for three weeks as micro-mass. The medium was replaced for twice a week, and later stained to identify differentiation.

Adipogenic differentiation of BMSCs

BMSCs were suspended in DMEM, and divided into six well plates. These were cultured for 48 hours. The cultured cells were grown for three additional days under 100% den-sity. Adipogenic induction medium [high glucose DMEM, 10% fetal bovine serum, 1 μM dexamethasone (Sigma), 10 ug/mL insulin (Sigma), 100 μM indomethacin (Sigma), and 0.5 mM methyl-isobutylzanthine (Sigma)] was used for a 72-hour culture and to induce differentiation. The medium was removed and replaced with adipogenic maintenance medium [high glucose DMEM, 10% fetal bovine serum, 10 ug/mL insulin (Sigma)] to culture for 24 hours. The re-placement of the media was repeated for three times. Then, finally, the sample was cultured for one additional week with adipogenic maintenance medium.

hematopoietic stem cells and MSCs can replace embryonic stem cells in protection and cell therapy of dying nerves.40-43

In fact, acquiring autologous stem cells, inducing less pain in patients, proliferating stem cells in vitro, differentiating, and transplanting cells back into the same patient is a new concept, and has interesting possibility as a new treatment method.

In this study, we used growth factors usually found in the human body to induce differentiation to neuron-like cells. We expect that this revolutionizing autologous cell therapy will be promising for the treatment of neurodegenerative diseases.

MATERIALS AND METHODS

Bone marrow collection and mesenchymal stem cell culture

Patients with no notable pathologic history were chosen for this study. Human bone marrow of three healthy donors aged 21 to 40 years was obtained from Institute of cell therapy center before 2005, after they gave informed consent accord-ing to the approved procedure. About 10 mL of marrow were collected from the pelvis of each patient and stored in a hepa-rin containing test tube. 30 mL of phosphate buffered saline (PBS) was added to marrow sample. Then, the mixture was slowly streamed down on Ficoll-Plaque TM plus solution. The mixture was centrifuged using density gradient centrifu-gation for 20 minutes in 2,000 rpm. Next, the top layer and monocyte layer present in the interface of Ficoll-Plaque TM plus were retrieved and mixed again with 20 mL of PBS. Again, the blend was centrifuged in 1,800 rpm for 5 minutes, and this time only the monocyte was retrieved.

The separated monocytes were placed in Dulbecco’s Mod-ified Eagle’s Medium (DMEM, 10 % FBS, 1×penicillin-streptomycin). Then, appropriate amounts of monocytes (2×105) were inoculated into a culture dish (T-75), and

cul-tured in an incubator.

After seven to ten days, since only MSCs in monocytes proliferated on the floor of the culture dish, the medium was replaced. Later, when MSCs proliferated over 80% of the culture dish, it was transferred to other culture dish (T-175) for subculture. The medium was replaced once every three to four days to subculture 5 times.

Fluorescence Activated Cell Sorting (FACS) analysis

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(SE-ville, MD, USA), HGF, 20 ng/mL, (R&D Systems, Minne-apolis, MN, USA), VEGF, 20 ng/mL, (R&D Systems), and was cultured with replacement of the medium at 3 day in-tervals for 14 days. The group with chemicals was cultured with the DMEM medium for seven days, and on the eighth day, a chemical compound (BHA 200 µM, KCl 5 mM, Val-proic acid 2 µM, Forskolin 10 µM, Hydrocortisone 1 µM, Insulin 5 µM) was added to the medium for an additional five days of culture.

Immunostaining

Immunochemical staining

The differentiated cells were suspended on a cm2 cover

slide at a 1×104 cells/cm2 concentration. After fixation, the

slide was washed with PBS for 5 minutes and fixed with 4% paraformaldehyde with PBS for 15 minutes. It was then washed twice with PBS for 5 minutes. Later, the sample was processed with PBS containing 1% BSA and 0.2% Tri-ton X-100 for 5 minutes. Primary antibody was mixed and allowed to react for 16 hours. Anti-human neuron-specific enolase (NSE); Chemicon Inc., Billerica, MA, USA), anti-human NeuN (Chemicon Inc.), anti-anti-human β-tubulin III (Sigma Co., St. Louis, MO, USA), anti-human glial fibril-lary acidic protein (GFAP); Sigma Co.), and anti-human microtubule-associated protein-2 (MAP-2) were used for primary antibodies. After the reaction with the primary anti-body, PBS with 0.5% BSA was used to wash twice for 15 minutes. The sample was cultured again with secondary an-tibody for 30 minutes. PBS with 0.5% BSA was used to wash twice for 5 minutes. Afterward, the Avidin-biotin re-action (Vectastain Elite ABC kit; Vector Laboratory Inc., Burlingame, CA, USA) was processed for 30 minutes. It was washed twice for 5 minutes with PBS. 3,3’-diamino-benzidine tetrahydrochloride dehydrate (DAB, Sigma Co.) was mixed as a staining substrate and left to react for five minutes. Processing with PBS for five minutes stopped the reaction and it was further washed with PBS two more times. The sample was dried and then washed with distilled water for five minutes. Finally, it was washed and dehydrat-ed with distilldehydrat-ed water, 70%, 80%, 95%, and 100% ethanol (in that order), and suspended.

Immunofluorescence staining

For immunofluorescent staining, the cells were transferred to a culture slide (four-chamber, BD Falcon, Franklin Lakes, NJ, USA) and cultured for 1-2 days. Using 4%

para-Confirmation of differentiation capability

Alkaline phosphatase staining of osteoblasts

Alkaline phosphatase staining method was used to confirm the differentiation of BMSCs into osteoblasts. Cells that had been cultured in a 24-well plate for three weeks were removed from the media and washed twice with PBS. Ice-cold methanol (99.9%) was added and fixed in room tem-perature for two minutes. Then, it was washed with tertiary distilled water. BCIP/NBP (Sigma) liquid substrate was add-ed and reactadd-ed with the sample for 10 minutes at an ambi-ent temperature. Again, the mixture was washed twice with tertiary distilled water. The stain results were later analyzed under an optical microscope.

Safranin O staining of chondrocytes

The cultured cells were suspended in ice-cold acetone for 1-2 minutes after washing PBS. Mayer’s hematoxylin solu-tion was used to stain for 10 minutes and washed with a bluing solution, and then with distilled water. 0.1% safranin O solution was added and allowed to react for five minutes. Every two minutes, the sample was added to the following solutions in order: 95% ethyl alcohol, absolute ethyl alco-hol, and xylene. The sample was mounted on a slide and their differentiation was studied under a microscope.

Oil red-O staining of adipocytes

Lipid staining using oil red O was used to confirm the dif-ferentiation of adipocytes from BMSCs. Cells that were cultured for 4 weeks in a six-well plate were mixed with 4% paraformaldehyde (in PBS) and fixed for 4 to 12 hours. The sample was then washed with 60% isopropanol (in PBS). It was stained with a 60% oil red-O solution (in PBS) for 45 minutes and washed with distilled water. The sample was tested under an optical microscope.

Neuron-like cell differentiation

The study was divided into two groups. One group was treat-ed with only growth factors, and the other was processtreat-ed with chemicals. First, BMSCs were cultured to occupy the entire culture medium and separated with a cell scraper. Masses of separated MSCs were then cultured to form 1-2 cluster/cm2. After 24 hours, when the BMSCs attached and

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Rock-10 minutes at 55- 60°C.

cDNA synthesis

The total RNA that had been separated was treated with DN-ase. RNA 1 ug was added to Cyclescript RT preMix (dT20, Bioneer Inc.) and cycle in Table 1 was repeated for 15 times to synthesize cDNA.

PCR

Using each synthesized cDNA as a mold, PCR was per-formed with the primers mentioned in Table 2. A total of 30 cycles were repeated.

Electrophoresis

Electrophoresis was performed on the DNA fragments syn-thesized by PCR using 1.5% agarose gel on 1X TAE buffer under 50 V. It was stained with ethidium bromide and ob-served under a U.V. illuminator.

Western blotting

Using a cell scraper, the cells in the medium were separated after being washed twice with PBS. After being collected into a 1.5 mL tube, radioimmunoprecipitation assay(RIPA) buffer [1% triton X-100, 1% sodium deoxycholate, 50 mM NaCl2, 50 mM tris-HCl, 1 mM sodium vanadate, 2 mM phenylmethanesulfonylfluoride (PMSF)] was added to ob-tain lysate. The protein sample was quantified with a protein assay solution, and was next added to 4× sampling buffer. The sample was boiled for five minutes. After electrophore-sis, transfer buffer (25 mM Tris base, 0.2 M glycine, 20% methanol) was used to transfer for one hour under 30V us-ing Immobilon membrane (Millipore). The sample was then treated with blocking buffer (5% skim milk, 1X TBS, 0.1% formaldehyde in PBS, cells were fixed for 4-8 hours and

washed three times with PBS. The sample was then added to blocking solution (5% BSA in PBS) and left to react for 1 hour. Primary antibody was allowed to react for one hour at 4°C. After washing with PBS three times, mounting so-lution was added and covered with a cover glass. The cells were observed under a fluorescent microscope. To test for expression of antigens of neurons, immunofluorescent stain-ing was performed usstain-ing Neuronal Nuclei (NeuN, 1 : 100, Chemicon Inc.), GFAP (1 : 1,000, Chemicon Inc.), MAP-2 (1 : 500, Chemicon Inc.), Neuron Specific Enolase (NSE, 1 : 100, Chemicon Inc.), TH2 (1 : 1,000, Chemicon Inc.), Gal C (1 : 500, Chemicon Inc.) as monoclonal antibodies.

RT-PCR

Total RNA separation

Each cell from 175 mm culture dish was washed twice with PBS. Then, 1 mL of AccuZol (Bioneer Inc., Alameda, CA, USA) was added and cells were harvested into 1.5 mL cen-trifuge tube using a cell scraper. After adding 200 µL of chlo-roform and shaking vigorously for 15 seconds, the mixture was left in ice for five minutes. The mixture was next centri-fuged for 15 minutes at 12,000 rpm at 4°C. After the super-natant was transferred to a 1.5 mL tube, an equal amount of isopropyl alcohol was added, and the mixture was left at -20°C for 10 minutes. At 4°C the mixture was centrifuged again for 15 minutes at 12,000 rpm. The supernatant was re-moved and was washed with 1 mL of 80% ethanol. The so-lution was centrifuged once again for 10 minutes in 12,000 rpm at 4°C, and the supernatant was removed. The ribonu-cleic acid (RNA) pellet was dried at room temperature for 10 to 20 minutes. It was dissolved into DEPC-water and left for

Table 1. cDNA Synthesis

Step Reaction Temperature Time

Step 1 Primer annealing 37°C 30 sec

Step 2 cDNA synthesis 48°C 4 min

Step 3 Melting secondary structure & cDNA synthesis 55°C 30 sec

Table 2. Prime Sequence for PCR

Primer sequence

GFAP for: GTG GGC AGG TGG GAG CTT GAT TCT rev: CTG GGG CGG CCT GGT ATG ACA NSE for: CCC ACT GAT CCT TCC CGA TAC AT rev: CCG ATC TGG TTG ACC TTG AGC A

Map2 for: CCA TTT GCA ACA GGA AGA CAC rev: CAG CTC AAA TGC TTT GCA ACT AT

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RESULTS

Marrow collection and MSCs culture

After seven days of culturing marrow derived eukaryocytes in the medium, cell clusters were confirmed under a micro-scope. The attached cells were forming typical mesenchy-mal stem cell shapes (form of neuroglial cells in a pyramid shape with many processes). The primary subculture was completed 15 days later (Fig. 1).

FACS analysis

FACS analysis was performed to confirm bone marrow-de-rived MSCs. Cells from the fourth subculture were collect-ed and testcollect-ed for SH2 and SH4 expressions, which are markers specific to mesenchymal stem cells. The test re-sults were 99.8% positive in cell culture. The test was nega-tive for antibodies (CD45, CD34, CD14), which are specif-ic markers to hematopoietspecif-ic stem cells, in the negative control group. Thus, it was confirmed that the sample of cells that had been grown consisted purely of mesenchymal stem cells, with no hematopoietic cells (Fig. 2).

Differentiation capability test

Alkaline phosphatase and silver nitrate staining of osteoblasts

When replaced with and cultured under high-glucose DMEM [10% fetal bovine serum, 0.1 µM dexamethasone (Sigma), 10 mM β-glycerol phosphate, 50 µM L-ascorbic acid] for three weeks, the stain for alkaline phosphatase and silver nitrate was positive. This confirmed the possibility of mes-enchymal stem cells’ differentiation to osteoblasts (Fig. 3).

Safranin O staininig of chondrogenic cells

After forming BMSCs into a micromass shape using a cen-trifuge, the cells were cultured in chondrogenic medium for three weeks. As a result of Safranin O stain, differentiation into chondrocytes was observed (Fig. 4).

Oil red-O staining of adipocytes

After culturing with adipogenic induction medium, the re-sult of Oil red-O staining showed and confirmed adipocyte differentiation (red fat vacuoles stained with Oil red-O in cells) (Fig. 5).

Differentiation into neuron-like cells

When cultured with the addition of growth factors or chem-TWEEN 20, polyoxyethylene (20) sorbitan monolaurate) for

one hour. At 4°C, antibody [GFAP, NSE, galactocerebroside (Gal C), and NeuN] was allowed to react for 12 hours. Then, washing buffer [1X TBS, 0.1% TWEEN 20, poly-oxyethylene (20) sorbitan monolaurate)] was used three times for five minutes each. Horseradish peroxidase bound secondary antibody was added to the reaction for one hour. It was washed three times with washing buffer. The result was verified with enhanced chemiluminescence (ECL) sys-tem (Habersham Bioscience).

Fig. 1. Mesenchymal stem cell culture (×100). (A) After 7 days of culturing. (B) After 14 days of culturing. (C) Subculture (4 times). (D) Cluster of BMSCs.

A

C

B

D

Fig. 3. Possibility of mesenchymal stem cells’ differentiation to osteoblasts (×100). (A) Alkaline phosphatese staining. (B) Silver nitrate staining.

A

B

Fig. 2. Fluorescence Activated Cell Sorting (FACS) analysis.

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100 101 102 103 104

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with for two weeks. When the grown and proliferated cells were immunohistochemically tested for neurocyte markers such as NSE, NeuN, and GFAP, the number of positive cells for each stain was very small in the group that was treated with EGF alone. However, when treated with both EGF and HGF, cells that showed positive stains for each staining method were clearly observed. Based on the results mentioned above, the medium treated with both EGF and HGF was much higher in positive immunohistochemical stain ratio than the medium processed with EGF only when investigated regarding the im-munohistochemical staining ratio that was positively stained in each condition (Table 3).

icals, each group showed that, as differentiation progressed, the cells’ processes became thinner and longer, similar to neurocytes, and morphologic changes were observed, such as the area of cytoplasm around the nucleus growing small-er. For the group with growth factor, the shape of neuron-like cells was observable a week later, and two weeks later, most of the cells were similar in shape to neurocytes. Particu-larly, in the group with chemical additions, filament struc-tures of various shapes were seen among the cells (Fig. 6).

Next, to compare and to observe the effects of growth fac-tors used in differentiation into neuron-like cells, EGF alone or a combination of EGF and HGF was given, to be followed up Fig. 5. Oil red-O staining of adipocytes. MSC, mesenchymal stem cell.

Control MSCs Adipocytes

Table 3. Immunohistochemically Tested for Neurocyte Markers

NSE NeuN GFAP

EGF 0.9 % 0.8 % 1.2 %

EGF and HGF 56 % 75 % 24 %

GFAP, glial fibrillary acidic protein; NSE, neuron specific enolase; NeuN, neuronal nuclei; EGF, epidermal growth factor; HGF, hepatocyte growth factor.

Control 1 wk

TGF 1 wk

Control 2 wk

TGF 2 wk

Control 3 wk

TGF 3 wk

Fig. 4. Cultured cells in chondrogenic medium for 3 weeks (×100). TGF, tumor growth factor.

Fig. 6. Differentiation into bone marrow-derived neuron-like cells (×200). (A) Mesenchymal stem cells. (B) Growth factor group (1 week). (C) Growth factor group (2 weeks). (D) Chemical group (2 weeks).

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godendrocyte was a strong posititve only in the group with growth factors. Through these results, we discovered that MSCs are capable of differentiating into neurocytes and glial cells; in addition, we could also confirm the differenti-ation of part of MSCs populdifferenti-ation into oligodendrocytes (Fig. 8).

RT-PCR

Although all the groups showed expression of the gene in the case of GFAP, the group with growth factor only showed increased expression of the gene. For NSE, the control group was expressed minutely. NSE was much more highly ex-pressed in the group with growth factor only. The group treated with chemicals also showed higher expression than the control group, but less than the group treated with growth factor. It was discovered that MAP2, NF-M, and GAP43 were only expressed in the group with chemical mediators. Based on these results, according to particular differentia-tion condidifferentia-tions, specific markers of some neuron-like cells show differences in expression level (Fig. 9).

Immunostaining

Immunochemical staining

For the group treated with growth factors for two weeks, im-munohistochemical staining of each neurocyte-specific mark-er (NSE, NeuN, and GFAP) was pmark-erformed. It was confirmed that all the markers were positively stained. Namely, it was verified that inducing MSCs differentiation into neuron-like cells by growth factors caused the cells to differentiate most-ly to neurocytes and glial cells (Fig. 7).

Immunofluorescence staining

In the case of immunofluorescent staining in glial fibrillary acidic protein (GFAP), the group treated with growth fac-tors for two weeks showed positive results. In the case of NeuN, the group with growth factors also resulted in posi-tive reactions and even the staining of the control group showed weakly fluorescent results. In the case of Microtu-bule-associated protein 2 (MAP2), the group with growth factors was a strong positive. Gal C that is a marker of

oli-Fig. 8. Immunofluorescence staining (×200). (A and B) GFAP, glial fibrillary acidic protein. (C and D) NeuN, neuronal nuclei. (E and F) MAP2, microtubule-associated protein 2. (G and H) Gal C, galactocerebroside.

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Fig. 7. Immunochemical staining (×200). (A) NSE, neuron specific enolase. (B) NeuN, neuronal nuclei. (C) GFAP, glial fibrillary acidic protein.

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BMSCs using growth factors such as EGF, VEGF, and HGF, we can presume the following three possibilities.

First, there exists a neurocyte subpopulation that can be differentiated into neurocytes in BMSCs that are proliferat-ed and culturproliferat-ed ex vivo. Second, BMSCs can be trans-dif-ferentiated using a method such as mesenchymal epithelial transformation (MET). Finally, as we already know from the results of the RT-PCR and western blotting, under par-ticular conditions of differentiation, it is possible to for cells to differentiate into neuron-like cells, each with different characteristics.

Bossolasco, et al.44 postulated that, since nestin and beta

tubulin III, together with O4 and GFAP showed a positive reaction, MSCs already have a subpopulation that has a ca-pability to differentiate into neuron-like cells. In addition, Ji-ang, et al.45 reported that selectively proliferated cells can be

differentiated into multipotent precursor cells with the char-acteristic shapes of neurocytes, astrocytes, and oligodendro-cytes. Chamberlain, et al.46 also proved the possible

trans-differentiation of mesenchymal stem cells.

Western blotting

With the exception of the control group, all the groups in-duced differentiation showed expression of the protein GFAP. The group with growth factor showed markedly higher ex-pression of protein than the group with chemicals. For NSE, the entire group showed expression of the protein, and the expression was particularly higher in the group with growth factor. In the Gal C test, the control group re-sults were weakly positive for Gal C expression, and higher than the expression found in the group with chemicals. NeuN was expressed in high quantity in both groups with growth factor and with chemicals. Based on these results, according to particular differentiation conditions, specific markers of some neuron-like cells show a difference in the level of expression (Fig. 10).

DISCUSSION

Through the results of neuron-like cell differentiation from

GFAP

NSE

Gal C

NeuN

Control group Growth factors 1 week group Growth factors 2 week group Chemical group

Fig. 10. Western blotting. GFAP, glial fibrillary acidic protein; NSE, neuron specific enolase; NeuN, neuronal nuclei; Gal C, galactocerebroside.

GFAP

NSE

MAP2

NF-M

GAP 43

Control group Chemical group Growth factors 1 week group Growth factors 2 week group

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mal cerebral infarct models, symptoms has resulted in re-covery and decreased neurological deficit when injected with MSCs.8,61-63 It has also been discovered that when

BM-SCs are injected into blood vessels, rather than directly into damaged brain tissue, the cells have shown an ability to pass through the blood-brain barrier and to migrate to the damaged part of the brain.2,20,21,61,64 In a clinical study, Bang,

et al.65 proliferated and introduced autologous MSCs

intra-venously to patients with cerebral infarction. They reported that injecting these stem cells had the effect of improving neurological symptoms, such as reducing the size of the in-farct and decreasing the atrophy of the ventricle.66

In this study, BMSCs’ ability to proliferate and differenti-ate into neuron-like cells by has indicdifferenti-ated a possibility for application to cell therapy for a wide range of diseases such as neurodegenerative disease and cerebral infarct. Previous-ly known methods of inducing neuron-like cell differentia-tion using several chemicals have limits because the toxici-ty of these chemicals makes them too risky to be applied in humans. However, the EGF, VEGF, and HGF used in this study are secreted in human body, and can be safely manip-ulated to induce differentiation. Furthermore, the use of dif-ferentiated neuron-like cells in clinical therapeutic methods may have fewer limitations. These cells also have autolo-gous marrow origin, and are thus free from severe compli-cations; for example, immune rejection reaction. Because large quantities of neuron-like cells can be acquired from a small amount of bone marrow, these cells’ application in clinical therapy will be very useful.

In the future, it will be necessary to compare and analyze the characteristics of neuron-like cells originating from MSCs with other neurons in molecular biological and phys-iological aspects. Further studies are required to uncover the possibilities of transplanting them into humans and differen-tiating the cells within animal.

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Currently, surgical and medical treatments for central nervous system diseases, including degenerative, traumatic, and ischemic damage, are very limited, and it is difficult to expect recovery from nervous system damage. To regener-ate the damaged nervous tissues, a method called neuron transplantation is a raised possibility. A study that transplant-ed fetal dopaminergic neurons to an adult who was suffering from Parkinson’s disease showed positive results.34

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Figure

Fig. 2. Fluorescence Activated Cell Sorting (FACS) analysis.

Fig 2.

Fluorescence Activated Cell Sorting FACS analysis . View in document p.6
Fig. 1. Mesenchymal stem cell culture (×100). (A) After 7 days of culturing. (B) After 14 days of culturing

Fig 1.

Mesenchymal stem cell culture 100 A After 7 days of culturing B After 14 days of culturing. View in document p.6
Fig. 3. Possibility of mesenchymal stem cells’ differentiation to osteoblasts (×100). (A) Alkaline phosphatese staining

Fig 3.

Possibility of mesenchymal stem cells differentiation to osteoblasts 100 A Alkaline phosphatese staining. View in document p.6
Fig. 6. Differentiation into bone marrow-derived neuron-like cells (×200). (A) Mesenchymal stem cells

Fig 6.

Differentiation into bone marrow derived neuron like cells 200 A Mesenchymal stem cells. View in document p.7
Fig. 7. Immunochemical staining (×200). (A) NSE, neuron specific enolase. (B) NeuN, neuronal nuclei

Fig 7.

Immunochemical staining 200 A NSE neuron specific enolase B NeuN neuronal nuclei. View in document p.8
Fig. 8. Immunofluorescence staining (×200). (A and B) GFAP, glial fibrillary acidic protein

Fig 8.

Immunofluorescence staining 200 A and B GFAP glial fibrillary acidic protein. View in document p.8
Fig. 9. Reverse transcriptase-Polymerase Chain Reaction. GFAP, glial fibrillary acidic protein; NSE, Neuron Specific Enolase; MAP2, Microtubule-associated protein 2; NF-M, Neurofilament-Middle; GAP 43, Growth Associated Protein 43.

Fig 9.

Reverse transcriptase Polymerase Chain Reaction GFAP glial fibrillary acidic protein NSE Neuron Specific Enolase MAP2 Microtubule associated protein 2 NF M Neurofilament Middle GAP 43 Growth Associated Protein 43 . View in document p.9
Fig. 10. Western blotting. GFAP, glial fibrillary acidic protein; NSE, neuron specific enolase; NeuN, neuronal nuclei; Gal C, galactocerebroside.

Fig 10.

Western blotting GFAP glial fibrillary acidic protein NSE neuron specific enolase NeuN neuronal nuclei Gal C galactocerebroside . View in document p.9

References

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